scholarly journals Nuclear mRNA metabolism drives selective basket assembly on a subset of nuclear pores in budding yeast

2021 ◽  
Author(s):  
Pierre Bensidoun ◽  
Taylor Reiter ◽  
Ben Montpetit ◽  
Daniel Zenklusen ◽  
Marlene Oeffinger
Keyword(s):  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Protein Complexes ◽  
Budding Yeast ◽  
Nuclear Pore ◽  
Nuclear Pores ◽  
Mrna Metabolism ◽  
Leakage Factor ◽  
Data Points ◽  
Cleavage And Polyadenylation

To determine which transcripts should reach the cytoplasm for translation, eukaryotic cells have established mechanisms to regulate selective mRNA export through the nuclear pore complex (NPC). The nuclear basket, a substructure of the NPC protruding into the nucleoplasm, is thought to function as a stable platform where mRNA-protein complexes (mRNPs) are rearranged and undergo quality control (QC) prior to export, ensuring that only mature mRNAs reach the cytoplasm. Here, we use proteomic, genetic, live-cell, and single-molecule resolution microscopy approaches in budding yeast to demonstrate that baskets assemble only on a subset of NPCs and that basket formation is dependent on RNA polymerase II (Pol II) transcription and subsequent mRNP processing. Specifically, we observe that the cleavage and polyadenylation machinery, the poly(A)-binding protein Pab1, and pre-mRNA-leakage factor Pml39 are required for basket assembly. We further show that while all nuclear pores can bind Mlp1, baskets assemble only on a subset of nucleoplasmic NPCs and these basket-containing pores associate a distinct protein and RNA interactome. Taken together, our data points towards nuclear pore heterogeneity and an RNA-dependent mechanism for functionalization of nuclear pores in budding yeast through nuclear basket assembly.

scholarly journals In vivo analysis of the stability and transport of nuclear poly(A)+ RNA.

1994 ◽  
Vol 126 (4) ◽  
pp. 877-899 ◽  
Author(s):  
S Huang ◽  
T J Deerinck ◽  
M H Ellisman ◽  
D L Spector
Keyword(s):  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Stable Population ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nuclear Pores ◽  
Electron Microscopic ◽  
Interchromatin Granule Clusters ◽  
Interchromatin Granule ◽  
Pore Complexes

We have studied the distribution of poly(A)+ RNA in the mammalian cell nucleus and its transport through nuclear pores by fluorescence and electron microscopic in situ hybridization. Poly(A)+ RNA was detected in the nucleus as a speckled pattern which includes interchromatin granule clusters and perichromatin fibrils. When cells are fractionated by detergent and salt extraction as well as DNase I digestion, the majority of the nuclear poly(A)+ RNA was found to remain associated with the nonchromatin RNP-enriched fraction of the nucleus. After inhibition of RNA polymerase II transcription for 5-10 h, a stable population of poly(A)+ RNA remained in the nucleus and was reorganized into fewer and larger interchromatin granule clusters along with pre-mRNA splicing factors. This stable population of nuclear RNA may play an important role in nuclear function. Furthermore, we have observed that, in actively transcribing cells, the regions of poly(A)+ RNA which reached the nuclear pore complexes appeared as narrow concentrations of RNA suggesting a limited or directed pathway of movement. All of the observed nuclear pores contained poly(A)+ RNA staining suggesting that they are all capable of exporting RNA. In addition, we have directly visualized, for the first time in mammalian cells, the transport of poly(A)+ RNA through the nuclear pore complexes.

scholarly journals Symplekin, a Constitutive Protein of Karyo- and Cytoplasmic Particles Involved in mRNA Biogenesis in Xenopus laevisOocytes

2002 ◽  
Vol 13 (5) ◽  
pp. 1665-1676 ◽  
Author(s):  
Ilse Hofmann ◽  
Martina Schnölzer ◽  
Isabelle Kaufmann ◽  
Werner W. Franke
Keyword(s):  
Immunofluorescence Microscopy ◽  
Protein Complexes ◽  
Gradient Centrifugation ◽  
Stage Iv ◽  
Cell Junctions ◽  
Xenopus Laevis Oocytes ◽  
Mrna Metabolism ◽  
Egg Extracts ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor

Symplekin is a dual location protein that has been localized to the cytoplasmic plaques of tight junctions but also occurs in the form of interchromatin particles in the karyoplasm. Here we report the identification of two novel and major symplekin-containing protein complexes in both the karyo- and the cytoplasm of Xenopus laevis oocytes. Buffer-extractable fractions from the karyoplasm of stage IV–VI oocytes contain an 11S particle, prepared by immunoselection and sucrose gradient centrifugation, in which symplekin is associated with the subunits of the cleavage and polyadenylation specificity factor (CPSF). Moreover, in immunofluorescence microscopy nuclear symplekin colocalizes with protein CPSF-100 in the “Cajal bodies.” However, symplekin is also found in cytoplasmic extracts of enucleated oocytes and egg extracts, where it occurs in 11S as well as in ca. 65S particles, again in association with CPSF-100. This suggests that, in X. laevis oocytes, symplekin is possibly involved in both processes, 3′-end processing of pre-mRNA in the nucleus and regulated polyadenylation in the cytoplasm. We discuss the possible occurrence of similar symplekin-containing particles involved in mRNA metabolism in the nucleus and cytoplasm of other kinds of cells, also in comparison with the nuclear forms of other dual location proteins in nuclei and cell junctions.

scholarly journals A single molecule localization microscopy method for tissues reveals nonrandom nuclear pore distribution in Drosophila

2021 ◽  
Author(s):  
Jinmei Cheng ◽  
Edward S. Allgeyer ◽  
Jennifer H. Richens ◽  
Edo Dzafic ◽  
Amandine Palandri ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cultured Cells ◽  
Ectopic Expression ◽  
Nuclear Pore ◽  
Optical Sectioning ◽  
Nuclear Pores ◽  
High Background ◽  
Chromatin Organisation ◽  
Microscopy Method ◽  
Localisation Microscopy

Single Molecule Localisation Microscopy (SMLM) can provide nanoscale resolution in thin samples but has rarely been applied to tissues, because of high background from out of focus emitters and optical aberrations. Here we describe a line scanning microscope that provides optical sectioning for SMLM in tissues. Imaging endogenously-tagged nucleoporins and F-actin on this system using DNA- and peptide-PAINT routinely gives 30 nm resolution or better at depths greater than 20 µm. This revealed that the nuclear pores are nonrandomly distributed in most Drosophila tissues, in contrast to cultured cells. Lamin Dm0 shows a complementary localisation to the nuclear pores, suggesting that it corrals the pores. Furthermore, ectopic expression of the tissue-specific Lamin C distributes the nuclear pores more randomly, whereas lamin C mutants enhance nuclear pore clustering, particularly in muscle nuclei. Since nucleoporins interact with specific chromatin domains, nuclear pore clustering could regulate local chromatin organisation and contribute to the disease phenotypes caused by human Lamin A/C laminopathies.

scholarly journals A method for single molecule localization microscopy of tissues reveals nonrandom distribution of nuclear pores in Drosophila

2021 ◽  
Author(s):  
Jinmei Cheng ◽  
Edward S Allgeyer ◽  
Jennifer H Richens ◽  
Edo Dzafic ◽  
Amandine Palandri ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cultured Cells ◽  
Ectopic Expression ◽  
Nuclear Pore ◽  
Optical Sectioning ◽  
Nuclear Pores ◽  
High Background ◽  
Chromatin Organisation ◽  
Localisation Microscopy ◽  
Single Molecule Localization Microscopy

Single Molecule Localisation Microscopy (SMLM) can provide nanoscale resolution in thin samples but has rarely been applied to tissues, because of high background from out of focus emitters. Here we describe a line scanning microscope that provides optical sectioning for SMLM in tissues. Imaging endogenously-tagged nucleoporins and F-actin on this system using DNA- and peptide-PAINT routinely gives 30nm resolution or better at depths greater than 20 μm. This revealed that the nuclear pores are nonrandomly distributed in most Drosophila tissues, in contrast to cultured cells. Lamin Dm0 shows a complementary localisation to the nuclear pores, suggesting that it corrals the pores. Furthermore, ectopic expression of the tissue-specific Lamin C distributes the nuclear pores more randomly, whereas lamin C mutants enhance nuclear pore clustering, particularly in muscle nuclei. Since nucleoporins interact with specific chromatin domains, nuclear pore clustering could regulate chromatin organisation locally and contribute to the disease phenotypes caused by human Lamin A/C laminopathies.

scholarly journals CRISPRpas: Programmable regulation of alternative polyadenylation by dCas9

2021 ◽  
Author(s):  
Jihae Shin ◽  
Qingbao Ding ◽  
Erdene Baljinnyam ◽  
Ruijia Wang ◽  
Bin Tian
Keyword(s):  
Rna Polymerase Ii ◽  
Target Location ◽  
Alternative Polyadenylation ◽  
Biological Processes ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Mrna Metabolism ◽  
Cleavage And Polyadenylation ◽  
Endogenous Genes ◽  
Dna Strand

ABSTRACTWell over half of human mRNA genes produce alternative polyadenylation (APA) isoforms that differ in mRNA metabolism due to 3’ UTR size changes or have variable coding potentials when coupled with alternative splicing. Aberrant APA is implicated in a growing number of human diseases. A programmable tool for APA regulation, hence, would be instrumental for understanding how APA events impact biological processes. Here, using a catalytically dead Cas9 (dCas9), we developed a method, named CRISPRpas, to alter cleavage and polyadenylation site (PAS) usage in 3’ UTRs or introns. We present key features that facilitate CRISPRpas, including targeting DNA strand, distance between PAS and targeting sequence, and strength of the PAS. For intronic PAS, we additionally analyze strengths of 5’ splice site and target location in intron. Our analyses implicate a dynamic competition between PAS usage and nascent RNA decay when RNA polymerase II elongation is blocked. We show modulation of APA of multiple endogenous genes including a gene that contains a single nucleotide polymorphism (SNP) that affects APA in the human population. CRISPRpas expands the CRISPR toolkit for perturbation of gene expression.

scholarly journals Promoter- and RNA polymerase II–dependent hsp-16 gene association with nuclear pores in Caenorhabditis elegans

2013 ◽  
Vol 200 (5) ◽  
pp. 589-604 ◽  
Author(s):  
Sabine Rohner ◽  
Veronique Kalck ◽  
Xuefei Wang ◽  
Kohta Ikegami ◽  
Jason D. Lieb ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Super Resolution ◽  
Single Copy ◽  
Nuclear Pore ◽  
Nuclear Pores ◽  
Yeast Genes ◽  
Nascent Transcripts

Some inducible yeast genes relocate to nuclear pores upon activation, but the general relevance of this phenomenon has remained largely unexplored. Here we show that the bidirectional hsp-16.2/41 promoter interacts with the nuclear pore complex upon activation by heat shock in the nematode Caenorhabditis elegans. Direct pore association was confirmed by both super-resolution microscopy and chromatin immunoprecipitation. The hsp-16.2 promoter was sufficient to mediate perinuclear positioning under basal level conditions of expression, both in integrated transgenes carrying from 1 to 74 copies of the promoter and in a single-copy genomic insertion. Perinuclear localization of the uninduced gene depended on promoter elements essential for induction and required the heat-shock transcription factor HSF-1, RNA polymerase II, and ENY-2, a factor that binds both SAGA and the THO/TREX mRNA export complex. After induction, colocalization with nuclear pores increased significantly at the promoter and along the coding sequence, dependent on the same promoter-associated factors, including active RNA polymerase II, and correlated with nascent transcripts.

scholarly journals Ccr4-Not and TFIIS Function Cooperatively To Rescue Arrested RNA Polymerase II

2015 ◽  
Vol 35 (11) ◽  
pp. 1915-1925 ◽  
Author(s):  
Arnob Dutta ◽  
Vinod Babbarwal ◽  
Jianhua Fu ◽  
Deborah Brunke-Reese ◽  
Diane M. Libert ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Molecular Mechanisms ◽  
Protein Complexes ◽  
Elongation Factors ◽  
Genetic Studies ◽  
Content Type ◽  
Mrna Metabolism ◽  
N Terminus ◽  
Biochemical Analyses

Expression of the genome requires RNA polymerase II (RNAPII) to transcribe across many natural and unnatural barriers, and this transcription across barriers is facilitated by protein complexes called elongation factors (EFs). Genetic studies inSaccharomyces cerevisiaeyeast suggest that multiple EFs collaborate to assist RNAPII in completing the transcription of genes, but the molecular mechanisms of how they cooperate to promote elongation are not well understood. The Ccr4-Not complex participates in multiple steps of mRNA metabolism and has recently been shown to be an EF. Here we describe how Ccr4-Not and TFIIS cooperate to stimulate elongation. We find that Ccr4-Not and TFIIS mutations show synthetically enhanced phenotypes, and biochemical analyses indicate that Ccr4-Not and TFIIS work synergistically to reactivate arrested RNAPII. Ccr4-Not increases the recruitment of TFIIS into elongation complexes and enhances the cleavage of the displaced transcript in backtracked RNAPII. This is mediated by an interaction between Ccr4-Not and the N terminus of TFIIS. In addition to revealing insights into how these two elongation factors cooperate to promote RNAPII elongation, our study extends the growing body of evidence suggesting that the N terminus of TFIIS acts as a docking/interacting site that allows it to synergize with other EFs to promote RNAPII transcription.

The Formation, Structure, Distribution and Frequency of Nuclear Pores

1971 ◽  
Vol 29 ◽  
pp. 518-519
Author(s):  
G. G. Maul
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Membrane ◽  
Dynamic Structure ◽  
Nuclear Pore ◽  
Nuclear Pores ◽  
Filter Function ◽  
Etching Technique ◽  
Selective Filter ◽  
Membranous Part

The chromatin of eukaryotic cells is separated from the cytoplasm by a double membrane. One obvious structural specialization of the nuclear membrane is the presence of pores which have been implicated to facilitate the selective nucleocytoplasmic exchange of a variety of large molecules. Thus, the function of nuclear pores has mainly been regarded to be a passive one. Non-membranous diaphragms, radiating fibers, central rings, and other pore-associated structures were thought to play a role in the selective filter function of the nuclear pore complex. Evidence will be presented that suggests that the nuclear pore is a dynamic structure which is non-randomly distributed and can be formed during interphase, and that a close relationship exists between chromatin and the membranous part of the nuclear pore complex.Octagonality of the nuclear pore complex has been confirmed by a variety of techniques. Using the freeze-etching technique, it was possible to show that the membranous part of the pore complex has an eight-sided outline in human melanoma cells in vitro. Fibers which traverse the pore proper at its corners are continuous and indistinguishable from chromatin at the nucleoplasmic side, as seen in conventionally fixed and sectioned material. Chromatin can be seen in octagonal outline if serial sections are analyzed which are parallel but do not include nuclear membranes (Fig. 1). It is concluded that the shape of the pore rim is due to fibrous material traversing the pore, and may not have any functional significance. In many pores one can recognize a central ring with eight fibers radiating to the corners of the pore rim. Such a structural arrangement is also found to connect eight ribosomes at the nuclear membrane.

Sign in / Sign up

Export Citation Format

Share Document